The activated form of galactose oxidase from the fungus Dactylium dendroides contains a single divalent copper ion which is antiferromagnetically coupled to a protein-based free radical. Chemical oxidation of the apoenzyme generates the free radical which is localized on a covalently crosslinked tyrosine-cysteine residue. This species, together with a model radical generated by UVirradiation of o-methylthiocresol (MTC), has been studied by high frequency EPR spectroscopy (139.5 [unreadable]GHz/5 Tesla) in conjunction with molecular orbital calculations employing self-consistent local density functional (LDF) methods. The Zeeman interactions (g-values) determined from the high frequency spectra of the apogalactose oxidase and the MTC model radicals are remarkably similar and support the assignment of the protein radical to a sulfur-substituted tyrosyl moiety. Molecular orbital calculations accurately reflect the experimental data, including a reduction (relative to unsubstituted tyrosyl radicals) in the difference in g-values whose principal axes lie in the plane of the phenoxy ring. High frequency echo-detected EPR spectra of the apogalactose oxidase radical resolve hyperfine splittings assigned to two methylene protons of the tyrosine and/or cysteine residues. The EPR spectra and MO calculations are consistent with the radical spin density being localized on the tyrosine-cysteine moiety, rather than delocalized throughout an extended 7t-network involving a nearby tryptophan as had been previously suggested as a possible explanation for the stability of the radical species.